1. Field of the Invention
This invention relates generally to technology useful for in vitro polynucleotide delivery applications. In one aspect, methods for monitoring intracellular activity in real time are disclosed. More particularly, this invention relates to polynucleotides and labeled gene carriers which monitor transport within cells and gene expression using a fluorescence microscope equipped with a mercury lamp, objective lenses, excitation filter, and a high resolution digital camera. The technology is useful for in vitro delivery applications in mammals.
2. Description of the Related Art
There is a need for non-viral drug and gene delivery systems having desirable properties such as low immunogenicity, amenable to production on a relatively large scale, and which can be easily modified to provide a range of biological properties. See Mulligan, R. C. Science 260, 926-932 (1993); and Luo, et al. Nat. Biotechnol. 18, 33-37 (2000). A number of polycationic polymers and lipids have facilitated plasmid DNA transfection of cells. See Kircheiset al. Adv. Drug Deliver. Rev. 2001, 53, 341-358. It is believed that polymer-DNA complexes enter cells by an endocytotic pathway as illustrated in FIG. 1.
It is generally recognized that there are four barriers to transport of a biomolecule, such as a gene, into the cell. These are the cell membrane, endosome membrane, nuclear membrane and the release of the biomolecule from the carrier. In the case of a nucleic acid, the nucleic acid-carrier complex must first pass through the cell membrane (path 1, FIG. 1). When this is accomplished by endocytosis, the nucleic acid-carrier complex is then internalized. The carrier along with the nucleic acid-cargo is enveloped by the cell membrane by the formation of a pocket and the pocket is subsequently pinched off (path 2, FIG. 1). The result is a cell endosome, which is a large membrane-bound structure enclosing the nucleic acid cargo, and the carrier. The nucleic acid-carrier complex must then escape from the endosome membrane into the cytoplasm (path 4, FIG. 1), avoid enzyme degradation in the cytoplasm, and cross the nuclear membrane (path 5, FIG. 1). Once in the nucleus, the nucleic acid cargo must separate from the carrier. In general, anything designed to overcome one or more of the barriers described above may be considered a delivery enhancer.
In general, delivery enhancers fall into two categories. These are viral carrier systems and non-viral carrier systems. As human viruses have evolved ways to overcome the barriers to transport into the nucleus discussed above, viruses or viral components are useful in transport of nucleic acid into cells. One example of a viral component useful as a delivery enhancer is the hemagglutinin peptide (HA-peptide). This viral peptide facilitates transfer of biomolecules into cells by endosome disruption. At the acidic pH of the endosome, this protein causes release of the biomolecule and carrier into the cytosol.
Non-viral delivery enhancers may be either polymer-based or lipid-based. They are generally polycations which act to balance the negative charge of the nucleic acid. Polycationic polymers have shown significant promise as non-viral gene delivery enhancers due in part to their ability to condense DNA plasmids of unlimited size and to safety concerns with viral vectors. Examples include peptides with regions rich in basic amino acids such as oligo-lysine, oligo-arginine or a combination thereof and polyethylenimine (PEI). These polycationic polymers facilitate transport by condensation of DNA. Branched chain versions of polycations such as PEI and Starburst dendrimers can mediate both DNA condensation and endosome release (Boussif, et al. (1995) Proc. Natl. Acad. Sci USA vol. 92: 7297-7301). PEI is a highly branched polymer with terminal amines that are ionizable at pH 6.9 and internal amines that are ionizable at pH 3.9 and because of this organization, can generate a change in vesicle pH that leads to vesicle swelling and eventually, release from endosome entrapment.
Another means to enhance delivery is to design a ligand on the carrier. The ligand must have a receptor on the cell that has been targeted for cargo delivery. Biomolecule delivery into the cell is then initiated by receptor recognition. When the ligand binds to its specific cell receptor, endocytosis is stimulated. Examples of ligands which have been used with various cell types to enhance biomolecule transport are galactose, transferrin, the glycoprotein asialoorosomucoid, adenovirus fiber, malaria circumsporozite protein, epidermal growth factor, human papilloma virus capsid, fibroblast growth factor and folic acid. In the case of the folate receptor, the bound ligand is internalized through a process termed potocytosis, where the receptor binds the ligand, the surrounding membrane closes off from the cell surface, and the internalized material then passes through the vesicular membrane into the cytoplasm (Gottschalk, et al. (1994) Gene Ther 1:185-191).
Various agents have been used for endosome disruption. Besides the HA-protein described above, defective-virus particles have also been used as endosomolytic agents (Cotten, et al. (July 1992) Proc. Natl. Acad. Sci. USA vol. 89: pages 6094-6098). Non-viral agents are either amphiphillic or lipid-based.
The release of biomolecules such as DNA into the cytoplasm of the cell can be enhanced by agents that either mediate endosome disruption, decrease degradation, or bypass this process all together. Chloroquine, which raises the endosomal pH, has been used to decrease the degradation of endocytosed material by inhibiting lysosomal hydrolytic enzymes (Wagner, et al. (1990) Proc Natl Acad Sci USA vol. 87: 3410-3414). Branched chain polycations such as PEI and starburst dendrimers also promote endosome release as discussed above.
To completely bypass endosomal degradation, subunits of toxins such as Diptheria toxin and Pseudomonas exotoxin have been utilized as components of chimeric proteins that can be incorporated into a gene/gene carrier complex (Uherek, et al. (1998) J. Biol. Chem. vol. 273: 8835-8841). These components promote shuttling of the nucleic acid through the endosomal membrane and back through the endoplasmic reticulum.
Once in the cytoplasm, the nucleic acid cargo must find its way to the nucleus. Localization to the nucleus may be enhanced by inclusion of a nuclear localization signal on the nucleic acid-carrier. A specific amino acid sequence that functions as a nuclear-localization signal (NLS) is used. The NLS on a cargo-carrier complex interacts with a specific nuclear transport receptor protein located in the cytosol. Once the cargo-carrier complex is assembled, the receptor protein in the complex is thought to make multiple contacts with nucleoporins, thereby transporting the complex through a nuclear pore. After a cargo-carrier complex reaches its destination, it dissociates, freeing the cargo and other components.
Technology that provides visualization of dynamic behaviors (paths 1-6, FIG. 1) of the complexes inside cells has been limited. Some workers have used electron microscopy to investigate the visualization of complexes inside cells. See Joshee et al. Human Gene Therapy 2002, 13, 1991-2004; and Panyam et al. Int. J. Pharm. 262, 1-11 (2003). However, this method does not allow the study of the dynamic behavior of living cells. Other workers have used confocal microscopy to study intracellular trafficking, but the system is expensive and observation could not be longer than 30 minutes. See Godbey, et al. PNAS 96, 5177-5181 (1999). Fluorescence microscopy has been used to observe a whole cell, but resolution inside the cell was poor. See Mathew et al. Gene Therapy, 10, 1105-1115 (2003).
In order to develop effective transfection reagents, there is a need for technology and methods to detect and monitor gene-DNA complexes inside cells. In this patent application, we disclosed technology and methods to monitor the real-time visualization and dynamic behavior inside cells.